LIPID PROFILE

DR.U.S.ARTHI
FIRST YEAR POST GRADUATE
 OBJECTIVES:
• Introduction
• Cholesterol
• Triglyceride
• Lipoprotein
• Low density lipoprotein
• High density lipoprotein
 INTRODUCTION:

• Plasma lipids consist of triacylglycerols (16%)

• Phospholipids(30%)
• Cholesterol (14%)
• Cholesteryl esters (36%) and
• Unesterified long-chain fatty acids(or FFAs) (4%)
CLASSIFICATION OF CLINICALLY
IMPORTANT LIPIDS
• Sterol Derivative: Cholesterol , Steroid hormones , Bile acids and
Vitamin D
• Fatty Acids: short chain ,Medium chain ,Long chain ,Prostaglandins
•
• Glyceryl Esters: Triglycerides, diglycerides, and monoglycerides
• Sphingosine Derivatives :Sphingomyelin ,Glycosphingolipids
• Terpenes (Isoprene Polymers): vitamin A,E and K
CHOLESTEROL:
TETRACYCLIC
PERHYDROCYCLOPENTANOPHENANTHRENE
SYNTHESIS:
Regulation

Insulin, thyroxine

sterol regulatory element-binding

protein (SREBP)
 ABSORPTION:

• Cholesterol ester is hydrolyzed by cholesterol-esterase ,the free

cholesterol is incorporated into bile salt micelle and absorbed into the
mucosal cell
•
• ABC (ATP binding cassette proteins) transporter proteins, ABCG5
(Sterolin 1) and ABCG8 (Sterolin 2) , limiting cholesterol absorption
Transport:
DRUGS:
• Statins act by inhibiting HMG-CoA reductase Examples are
atorvastatin, simvastatin, fluvastatin, and pravastatin

• Ezetimibe reduces blood cholesterol levels by inhibiting Neimann-

Pick C-like 1 protein(absorption)

• Fibrates such as clofibrate, gemfibrozil and nicotinic acid, which act

mainly to lower plasma triacylglycerol
 SAMPLE COLLECTION:

• Heparin or EDTA tube

• Storage
• 7 days :at 25 degree celcius/ 4-8 degree celcius
• 3 months: at – 20 degree celcius
Estimation by enzymatic method
 REFERENCE VALUE :

• Normal range:140-200 mg/dl

• Linearity up to 1000 mg/dL ,erba (695mg/dl)

• Measuring range:4-695mg/dl
Interfering substances:
• Bilirubin upto 20 mg/dL (D)(spectral property)
• Hemoglobin upto 5 g/l of plasma (I) (pseudo peroxidase)
• Triglycerides upto 2000mg/dl

• Free or unesterified cholesterol can be readily quantified by deleting

the cholesterol esterase from the reagent
• Cholesterol oxidase can react with sterols of patients with β-
sitosterolemia

• Ascorbic acid is a reducing agent compete with the chromogenic

substrates for hydrogen peroxide ,high ascorbic acid (30 mg/dl)can
result in lower measured levels of cholesterol
 ESTIMATION OF CHOLESTEROL:

• Definitive method for cholesterol is isotope dilution mass

spectrometry
•
• Chemical method:Abell-kendall method
TRIGLYCERIDE :
SYNTHESIS:
ABSORPTION:
 stryer

Perilipin regulates balance

between storage and
lipolysis
ESTIMATION BY ENZYMATIC(GPO)
METHOD:
• Linearity :1000mg/dl
• Normal range:50-150mg/dl

• No Interference bilirubin upto 20 mg/dl

hemoglobin upto 10 g/l
ESTIMATION

• Reference method uses a chloroform extraction procedure followed

by silicic acid chromatography(Adsorption) to isolate TG

Classical method(modifcation of van Handel)

• Glycerol is released by saponification (alkaline hydrolysis of
triglycerides) and is oxidized with sodium periodate:
Glycerol + NaIO4 Formaldehyde +Formic acid
TRIGLYCERIDE BLANK:
• Glycerol is normally present in plasma in concentrations below (1.5 mg/dL)
equivalent to a triglyceride of about 14 mg/dL

• High after exercise, uncontrolled diabetes, contamination with the glycerol

lubricant used on the stoppers, ingestion of glycerol-containing medications; or
in rare disorder, hyperglycerolemia

• Blank assay, without the addition of lipase, provides a measure of preexisting

glycerol and increase reading in blank indicate presence of glycerol
Lipoprotein
APOLIPOPROTEIN( PROTEIN COMPONENT OF LIPOPROTEIN)
CHYLOMICRONS:
• Main sites of metabolism of chylomicrons are adipose tissue and skeletal muscle
The half-life of chylomicrons in blood is about 1 hour
• The enzyme lipoprotein lipase (LpL) is located at the endothelial layer of
capillaries of adipose tissue, muscles and heart; but not in liver
• Apo C-II present in the chylomicrons activates the LpL
• The LpL hydrolyzes triglycerides present in chylomicrons into fatty acids and
glycerol
• Insulin increases LpL activity
VERY LOW DENSITY LIPOPROTEIN:

• They are synthesized in the liver from glycerol and fatty acids and
incorporated into VLDL along with hepatic cholesterol, apo-B-100,
C-II and E

• Half life 1 to 3 hours

• Function: Carry endogenous triglyceride from liver to peripheral
tissue
 VLDL-C/PLASMA TRIGLYCERIDE RATIO

• The ratio of VLDL-C to plasma triglycerides may be useful in the

evaluation of type 3 hyperlipoproteinemia
• This ratio, expressed in mol/mol or (mass/mass), is generally in the
range of 0.230 to 0.575 (0.1 to 0.25) in samples without β-VLDL
• Overt type 3 patient manifest both β-VLDL and a VLDL-C/plasma
triglyceride ratio of 0.689 (0.3) or greater
LOW DENSITY LIPOPROTEIN
• The liver and many extrahepatic tissues express the LDL(apo B-100,
E) receptor (LDL receptor)

• The LDL receptor is found in many tissues in the body, including the
liver, arterial wall, ovary, testes, and adrenocortex
Estimation of LDL by direct method:

Based on modified polyvinyl sulfonic acid(pvs) and polyethylene glycol

methyl ether(pegme) coupled classic precipitation method
• Normal range <100 mg/dl
• Linearity 263 mg/dl
• Measuring range:2.60 -263 mg/dl
• No Interference: hemoglobin upto 10g/l
• Bilirubin 20 mg/dl
• TGL 2000mg/dl
• The first, a reference laboratory procedure, involves ultracentrifugation
to separate LDL from other lipoproteins, followed by analysis
• Second method use the Friedewald formula to calculate LDL-C
• Finally, more recently developed homogeneous methods for measuring
LDL-C
 Friedewald equation :LDL cholesterol = total cholesterol – HDL
cholesterol – VLDL(total triglycerides/5)
• The Friedewald equation is inaccurate when the concentration of
triglycerides is high (>~400 mg/dL) or when there is an appreciable
number of chylomicrons

• Non-HDL cholesterol represents cholesterol in the lipoproteins that

contain beta-apolipoproteins (i.e., B-48 or B-100).
BETA QUANTIFICATION : REFERENCE METHOD

• Beta quantification assumes that virtually all cholesterol is contained in the

three major lipoprotein classes (i.e., VLDL, LDL, and HDL)
• Plasma is ultracentrifuged for at least 18 hours at 105k*g , VLDL and CMs
accumulate as a floating layer, leaving predominantly LDL and HDL in
solution

• Because TG-rich lipoproteins are removed from this sample, measurement of

LDL-C is more reflective of actual concentration
 HIGH DENSITY LIPOPROTEIN:
• The main receptor for taking up HDL is the Scavenger Receptor B1
SR-B1) in liver
• SR-B1 has a dual role in HDL metabolism acts as endogenous receptor
for HDLs in the liver
• HDL-SR-B1 interaction in the adrenal glands is the mechanism for the
delivery of cholesterol to the steroid hormone synthesizing cells of this
tissue
• The ABC-G1 transport of cholesterol from cells to HDL
• ABC-A1 helps efflux of cholesterol into apo-A1 in the discoid HDL
HDL Cycle
• HDL3 generated from discoidal HDL by the action of LCAT, accepts
cholesterol from the tissues via the SR-B1 and cholesterol is then esterified by
LCAT, increasing the size of the particles to form the less dense HDL2
•
• HDL3 is then reformed either after selective delivery of cholesteryl ester to the
liver via the SR-B1 or by hydrolysis of HDL2 phospholipids and triacyl
glycerol by “hepatic lipase”
FORMATION OF PRE-Β HDL

• Free apo-A1 is released by these processes and forms pre β-HDL

after associating with a minimum amount of phospholipids and
cholesterol
• Surplus apo-A1 is destroyed in the kidney
• Pre beta –HDL is potent inducer of cholesterol efflux
 Estimation by homogenous methods
Based on modified polyvinyl sulfonic acid(PVS) and polyethylene glycol methyl
ether(PEGME) coupled classic precipitation method

• LDL,VLDL and chylomicrons reacts with PVS and PEGME form inaccessible ldl,vldl and
chylomicrons by cholesterol oxidase and cholesterol esterase
ESTIMATION:
• Reference method: Combination of Ultra Centrifugation and Polyanion
precipitation to isolate HDL in this VLDL and chylomicrons if present
removed by ultra centrifugation, which floats on top remove by tube slicing
technique
• Apo B 100 lipoproteins are precipitated by adding heparin and manganese
chloride to water(precipitate agent)
• Reference range: Male 35-80mg/dl
Female: 40-88 mg/dl
Linearity:193 mg/dl
Measuring range:1.90-193 mg/dl
Interferences :
Free Haemoglobin upto 10 g/l , bilirubin upto 10 mg/dl ,
TGL upto 2000mg/dl
Drugs :NAC,metamizole and acetaminophen
Polyanion Precipitation Methods
• Some lipoproteins are precipitated with polyanions such as
heparin sulfate, dextran sulfate, phosphotungstate, and others in the
presence of divalent cations such as Ca++, Mg++, and Mn++
• It is easier to separate apoB-containing lipoproteins from HDL than
it is to separate VLDL from LDL or HDL2 from HDL3
• Laboratories have replaced precipitation techniques with automated
homogeneous assays for HDL-C and LDL-C
LIPOPROTEIN (A)

• Lp(a) inhibits fibrinolysis

• Levels more than 30 mg/dL increase the risk 3 times; and when
increased Lp(a) is associated with increased LDL, the risk is
increased 6 times
• Nicotinic acid will reduce serum Lp(a) level

• Lp (a)<0.30g/l
 LP(a)

• Lp(a) has a density similar to LDL, but it migrates similarly to VLDL

on electrophoresis, an additional band with pre-β mobility
• Lp(a) can now be measured using immunoturbidimetric methods
• To estimate the contribution of Lp(a)-cholesterol to the measured
LDL-C value, where the values are given in mg/dL
• Lp(a)cholesterol =Lp (a )mass × 0 .3
• LDL-C =TC –(HDL-cholesterol) -Plasma TG /5-0.3(Lp (a)mass)
 APOLIPOPROTEINS:

• Immunoassay(ELISA)
• Immunoturbidimetric and Immunonephelometry
STANDING PLASMA TEST
• Chylomicrons, if present in appreciable quantities, are detected using
the “standing plasma” test aliquot of plasma (2 mL) is placed in test
tube and allowed to stand in the refrigerator at 4° C undisturbed
overnight
• Chylomicrons accumulate as a floating “cream” layer and can be
detected visually which is considered to be abnormal
• A plasma sample that remains turbid after standing overnight contains
excessive amounts of VLDL; if a floating “cream” layer also forms,
chylomicrons are present as well
DISORDERS
HIGH TRIGLYCERIDES WITH NORMAL
CHOLESTEROL(TYPE 1)
• Diabetic dyslipidemia
• Familial hypertriglyceridemia
• Lipoprotein lipase deficiency (hyperchylomicronemia)
• Apo C-II deficiency
• Apo C-III excess
HIGH CHOLESTEROL WITH HIGH LDL-C(TYPE 2A)

• Familial hypercholesterolemia –LDLR gene defect

• Non familial hypercholesterolemia
• Defective Apo B 100
• Sitosterolemia( mutation of ABCG8 OR G5 gene) normal
range<1mg/dl
HIGH CHOLESTEROL WITH HIGH TRIGLYCERIDES(TYPE 2B
& 3)

• Familial Combined Hyperlipidemia (Type 2B)

• Acquired Combined Hyperlipidemia
• Dysbetalipoproteinemia (Type 3)(VLDL-C/TGL ratio > 0.3)
• Hepatic Lipase Deficiency(TC 250-1500 mg/dl and
TGL 400-8000mg/dl
• Cholesterol 7-Alpha-Hydroxylase Deficiency
REFERENCES:

• Tietz text book of clinical chemistry 6 th edition

• Harper 32 edition
• D M Vasudevan 10 th edition
• Henry’s clinical diagnosis and management by laboratory
methods 23 edition
• Lippincott 6 edition
• Kaplan 5 edition
THANK YOU